Mdr Xdr Tb What Microbiologist Should Know Iamm 2009


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Recent emergence of XDR TB have brought home the fact that reliable laboratory work up is essential if we are going to control tuberculosis.

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Mdr Xdr Tb What Microbiologist Should Know Iamm 2009

  1. 1. MDR / XDR TB What Microbiologists should know Dr. Ashok Rattan, Chief Executive, Fortis Clinical Research Ltd., Adviser, Religare SRL Diagnostics in Fortis / Escorts Hospitals in Delhi & NCR
  2. 2. Milestones in Laboratory Diagnosis of Tuberculosis <ul><li>Koch (1882) Alkaline methylene blue, vesuvin as counterstain </li></ul><ul><li>Ehrlich (1882) Discovered acid fastness, hot fuchsin in aniline oil </li></ul><ul><li>Ziehl (1883) Phenol as mordant </li></ul><ul><li>Neelsen (1884) Combined dye & mordant </li></ul><ul><li>Lehmann & </li></ul><ul><li>Neumann (1896) Myco bacteria </li></ul>
  3. 3. <ul><li>Story of anti-tuberculosis chemotherapy is a miniature history of anti-infective chemotherapy </li></ul><ul><li>First half of 20 th century </li></ul><ul><ul><li>Thick lipid rich wall of mycobacteria postulated to make chemotherapy impossible </li></ul></ul><ul><ul><li>Sulfonamides & Penicillin had no useful activity against MTb </li></ul></ul>
  4. 4. Prof Selman A Waksman & Albert Schatz Streptomyces griseus Streptomycin The drug would lead the path in the elimination of “The Great White Plague”
  5. 5. British Medical Research Council (BMRC) trial 1948 <ul><li>Investigated efficacy of streptomycin monotherapy </li></ul><ul><li>Most patients who were treated with the drug developed resistant strains </li></ul>
  6. 6. Jorgen Lehman para aminosalicyclic acid (PAS) 1943 Lehman J. On the effect of isomers of para aminosalicyclic acid And related substances on the tuberculostatic effect of PAS. Experientia 1949; 5: 365 – 62. Turnbull FW et al. Streptomycin resistance after treatment with PAS alone. BMJ 1953; 1: 1244 - 46
  7. 7. PAS helps prevent emergence of resistance to SM
  8. 8. INH, a pro drug with anti TB activity (1952) first synthesized in 1912 as MAO inhibitor
  9. 11. Second line anti TB drugs six different classes of drugs <ul><li>Category Drugs . </li></ul><ul><li>1. Aminoglycosides Kanamycin </li></ul><ul><li>Amikacin </li></ul><ul><li>2. Thioamides Ethionamide </li></ul><ul><li> Prothiomide </li></ul><ul><li>3. Polypeptide Capreomycin </li></ul><ul><li>4. FQ Ciprofloxacine </li></ul><ul><li>Ofloxacin </li></ul><ul><li>5. Cycloserine/ Terizidone </li></ul><ul><li>6. PAS </li></ul>
  10. 14. Resistance due to mutation in target gene <ul><li>Rifampicin 3.32 x 10 -9 </li></ul><ul><li>INH 2.56 x 10 -8 </li></ul><ul><li>Streptomycin 2.29 x 10 -8 </li></ul><ul><li>Ethambutol 1.0 x 10 -7 </li></ul><ul><ul><li>Risk of simultaneous </li></ul></ul><ul><ul><li>Resistance to two drugs = sum of mutational frequency </li></ul></ul><ul><ul><li>For Rif, SM, INH = 10 –25 </li></ul></ul><ul><ul><li>P = 1 – (1 - r) n </li></ul></ul><ul><ul><li>P= probability of drug resistance, r = mutation rate </li></ul></ul><ul><ul><li>N = number of bacilli in a lesion (usually 10 -- 8 ) </li></ul></ul>
  11. 15. rpo B gene mutations
  12. 16. Mitchisen Actively dividing mycobacteria mimicked by in vitro tests (aerobic environment) Myobacteria with spurts of metabolism Microaerophilic conditions Intracellular mycobacteria Acidic pH Dormant Mycobacteria Metabolic activity Rate of division INH RMP RMP PZA
  13. 17. The 5 Components of the DOTS Strategy: <ul><li>Sustained political commitment </li></ul><ul><li>Access to quality-assured sputum microscopy </li></ul><ul><li>Standardized short-course chemotherapy for all cases of TB under proper case management conditions, including Directly Observed Treatment (DOT) </li></ul><ul><li>Uninterrupted supply of quality-assured drugs </li></ul><ul><li>Recording and reporting system enabling outcome assessment of all patients and assessment of overall program performance </li></ul>
  14. 18. There is a gathering storm of drug resistant tuberculosis and the strategy of WHO and other national & international agencies has failed to control the disease or prevent emergence of MDR / XDR TB Doctors without borders MSF Alert Fall 2006
  15. 19. Tuberculosis now There is a gathering storm of drug resistant tuberculosis and the strategy of WHO, other national & international agencies has failed to control the disease or prevent emergence of MDR/XDR TB - Doctors without borders MSF Alert Fall 2006 500,000 MDR TB 2 million death 10 million cases DOTS detects 70% Cures 85% 2 billion persons With latent TB
  16. 20. MDR/XDR TB is Man Made <ul><li>MDR TB is a laboratory Diagnosis </li></ul><ul><li>Simultaneous resistance to INH and Rifampicin </li></ul><ul><li>Generated as a consequence of human error </li></ul><ul><ul><li>Patient, Prescriber or Health care system </li></ul></ul><ul><ul><ul><li>Prescription of drugs </li></ul></ul></ul><ul><ul><ul><li>Management of drug supply </li></ul></ul></ul><ul><ul><ul><li>Case management </li></ul></ul></ul><ul><ul><ul><li>Process of drug delivery to the patient </li></ul></ul></ul><ul><ul><ul><li>Compliance of the patient </li></ul></ul></ul><ul><ul><ul><li>Distance of healthcare centre </li></ul></ul></ul><ul><ul><ul><li>Lack of transport </li></ul></ul></ul>
  17. 21. MDR-TB Why worry ? <ul><ul><li>DOTS don't yield acceptable cure rates </li></ul></ul><ul><ul><li>Treatment is more expensive </li></ul></ul><ul><ul><ul><li>Treatment of susceptible strains $ 200 </li></ul></ul></ul><ul><ul><ul><li>Treatment of MDR infections $ 20,000 </li></ul></ul></ul><ul><ul><li>Without effective treatment, transmission will continue </li></ul></ul><ul><ul><li>Private tragedy, Public Health Emergency </li></ul></ul><ul><ul><li>Use of DOTS alone in MDR will lead to generation of XDR TB </li></ul></ul>
  18. 22. Contributing factors to Development of MDR/XDR TB <ul><li>Patient </li></ul><ul><ul><li>Poor adherence to medication </li></ul></ul><ul><li>Provider: </li></ul><ul><ul><li>Prescribing errors </li></ul></ul><ul><ul><li>Suboptimal dosing </li></ul></ul><ul><ul><li>Insufficient monitoring of therapy </li></ul></ul><ul><ul><li>Drug interactions in co-morbidities </li></ul></ul><ul><li>Healthcare system: </li></ul><ul><ul><li>Poorly functioning TB control program </li></ul></ul><ul><ul><li>Medication stock outs </li></ul></ul><ul><ul><li>Lack of staff </li></ul></ul><ul><ul><li>Inadequate access for patients: distance, transportation </li></ul></ul><ul><ul><li>Policies that rely on smear alone to diagnose TB </li></ul></ul>
  19. 23. Risk factors for drug resistant TB <ul><li>Residence in a region with known high MDR/XDR TB prevalence </li></ul><ul><li>Known exposure to MDR/XDR TB patients </li></ul><ul><li>HIV/AIDS </li></ul><ul><li>History of </li></ul><ul><ul><li>Previous TB therapy </li></ul></ul><ul><ul><li>Relapse or recurrence after previous successful treatment </li></ul></ul><ul><ul><li>Hospitalization within the past two years </li></ul></ul><ul><ul><li>Nonadherence or default </li></ul></ul><ul><ul><li>Incarceration </li></ul></ul><ul><ul><li>Substance abuse </li></ul></ul><ul><ul><li>Malaborsption </li></ul></ul><ul><ul><li>Medications known to cause drug interaction with ATT </li></ul></ul>
  20. 24. Predicting the future of XDR Tuberculosis Sally Blower & Virginie Supervie. Lancet 2007; 7: 443 <ul><li>WHO specified targets for reducing global tuberculosis epidemic </li></ul><ul><ul><li>Detecting & treating at least 70% of sputum smear positive cases (currently 53% are detected) </li></ul></ul><ul><ul><li>Attaining 85% cure rate (currently 82% of drug sensitive cases are cured </li></ul></ul><ul><ul><li>Epidemics that are driven mainly by drug sensitive strains my be controlled by achieving WHO targets (2005) </li></ul></ul>
  21. 25. Predicting the future of XDR Tuberculosis Sally Blower & Virginie Supervie. Lancet 2007; 7: 443 <ul><li>In South Korea & Latvia: 15 – 19% MDR </li></ul>Without effective control of MDR TB epidemics  XDR TB would become uncontrollable
  22. 26. Pillay & Sturm. CID 2007; 45: 1407- 14 <ul><li>Possible Reason for conversion of MDR to XDR within a decade </li></ul><ul><li>Introduction of DOTS & DOTS + in TB Control </li></ul><ul><li>No susceptibility testing or Drug Susceptibility Surveillance </li></ul>
  23. 27. Venn diagram of SNP Jassal & Bishoi: Lancet Infect Dis 2009; 9: 10 - 30
  24. 28. <ul><li>Basis of TB drug resistance is selection of bacterial mutants with innate resistance to chemotherapy </li></ul><ul><li>Generated by three inter related mechanisms </li></ul><ul><ul><li>Conversion of wild type pan sensitive strain to resistant strain during therapy (acquired resistance) </li></ul></ul><ul><ul><li>Increased development of resistance in drug resistant strains because of inappropriate chemotherapy (amplified resistance) </li></ul></ul><ul><ul><li>Transmission of drug resistant cases (transmitted resistance) [fuelled by HIV epidemic] </li></ul></ul>
  25. 29. Generation of resistance with monotherapy Susceptible Resistant (SM)
  26. 30. Generation of resistance with monotherapy Susceptible Resistant (SM R) (ETH R) ETH
  27. 31. 50 countries
  28. 32. What is XDR TB Laboratory Based Diagnosis <ul><li>March 2006: MMWR(2000 – 2004) </li></ul><ul><ul><li>INH+ RIF (MDR) + R to at least 3 of 6 classes of second line agents : </li></ul></ul><ul><ul><ul><ul><li>Aminoglycosides; </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Polypeptides; </li></ul></ul></ul></ul><ul><ul><ul><ul><li>FQ; </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Thioamides; </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Cycloserine; </li></ul></ul></ul></ul><ul><ul><ul><ul><li>PAS </li></ul></ul></ul></ul><ul><li>WHO Task Force Oct 2006 </li></ul><ul><ul><ul><ul><li>MDR + FQ + any one of Amikacin/ Kanamycin/ Capreomycin, </li></ul></ul></ul></ul>Bactericidal & reliable in vitro methods available
  29. 33. Is the current definition of XDR TB adequate ? <ul><ul><ul><li>Is XDR TB predictive of poorer clinical response ? </li></ul></ul></ul><ul><ul><ul><ul><li>Success rate of >90% achieved with pza+ethionamide+cycloserine </li></ul></ul></ul></ul><ul><ul><li>Most accurate diagnosis of XDR TB would be: </li></ul></ul><ul><ul><ul><li>R to all first line drugs (not only INH+Rif) </li></ul></ul></ul><ul><ul><ul><li>+ FQ (Moxifloxacin) </li></ul></ul></ul><ul><ul><ul><li>+ all 3 injectables (not just to one) </li></ul></ul></ul><ul><ul><ul><ul><li>These cases will have < 50% chances of success & so would behave differently from other XDR TB strains </li></ul></ul></ul></ul><ul><ul><li>If not, </li></ul></ul><ul><ul><ul><li>we would soon also be talking about XXDR (extensively extensively drug resistant TB) or </li></ul></ul></ul><ul><ul><ul><li>CDR TB (completely drug resistant) </li></ul></ul></ul>
  30. 34. M. tuberculosis Drug susceptibile XDR M. tuberculosis Drug susceptible INH + RIF Resistant = MDR FQ Resistant Injectable Resistant
  31. 35. 1 st line drugs 2 nd line drugs INH INH RIF RIF PZA PZA Etham Etham FQ FQ Injectable Injectable Ethio Ethio Cyclo Cyclo PAS PAS MDR TB XDR TB 2 most important 2 most important
  32. 36. <ul><li>Preventing transmission of M tuberculosis relies on </li></ul><ul><ul><li>Accurate & Rapid diagnosis </li></ul></ul><ul><ul><li>PT results TAT </li></ul></ul><ul><ul><li>Key barrier </li></ul></ul><ul><ul><ul><li>Current case detection rates are low </li></ul></ul></ul><ul><ul><ul><li>Methods insensitive & slow </li></ul></ul></ul><ul><ul><li>Delays enhance likelihood of transmission </li></ul></ul><ul><ul><li>Potential to produce clusters of secondary infections </li></ul></ul><ul><ul><ul><li>Transmission in nosocomial or congregate settings </li></ul></ul></ul>
  33. 37. Commonest method for diagnosis of TB 1885 2005
  34. 38. <ul><li>AFB smear </li></ul><ul><li>Culture on LJ </li></ul><ul><li>Identification by Niacin & </li></ul><ul><li>Nitrate test </li></ul><ul><li>Sensitivity by RR </li></ul><ul><li>Culture report in 8wk </li></ul><ul><li>DST report in 8 wks </li></ul><ul><li>Treatment duration from 9 M to 18 M </li></ul>Slow diagnostic methods
  35. 39. Progress made in laboratory diagnosis of tuberculosis <ul><li>Fluorochrome stain for AFB smear Mid 1970s </li></ul><ul><li>BACTEC 460TB Early 1980s </li></ul><ul><li>DNA probes for AFB +ve respiratory Late 1980s </li></ul><ul><li>Automated broth systems for culture Early 1990s </li></ul><ul><li>HPLC on AFB +ve cultures Early 1990s </li></ul><ul><li>Drug susceptibility by Bactec Early 1990s </li></ul><ul><li>Nucleic acid amplification on all smear +ve 1996 </li></ul><ul><li>Use of courier to transport specimen 1997 </li></ul><ul><li>Us of NAA TB testing on smear –ve 2000 </li></ul><ul><li>Reporting of results by fax 2000 </li></ul><ul><li>Lipa for identification and Rif resistance 1997 </li></ul><ul><li>Genotype MTBDR for subspecies 2003 </li></ul>
  36. 40. Think Globally Act locally
  37. 50. Sample & culture positive load for AFB Bactec at GGN (September 2007-October 2008) sample number = 1656
  38. 51. Total samples processed = 1656 (smear +ve=302, -ve=1354) Total culture positive =453 (Mtb=393, MOTT=60) 30.62 13.79 8 41 MOTT Smear Negative (n=1354) 34.28 17.15 66 133 M.tb 28 6.6 10 21 MOTT 26.12 9.64 191 258 M.tb Smear Positive (n=302) Speed of growth on LJ in days Speed of growth on MGIT in days LJ +ve MGIT +ve Mycobacteria spp. Smear Result
  39. 52. Speed Of Growth Of M.tb (MGIT 960 vs LJ Media) 1 st wk 2 nd wk 3 rd wk 4 th wk 5 th wk 6 th wk 166 0 132 23 55 46 23 63 13 48 2 24
  40. 53. Policy guidance on DST of second line anti tuberculosis drugs : WHO 2008 <ul><li>Alternative method for grouping ATT drugs </li></ul><ul><li>Grouping Drugs </li></ul><ul><ul><li>Group 1 </li></ul></ul><ul><ul><ul><li>First line oral drugs: INH (H); rifampicin (R), ethambutol (E), pyrazinamide (Z), rifabutin (Rib) </li></ul></ul></ul><ul><ul><li>Group 2 </li></ul></ul><ul><ul><ul><li>Injectable agents Kanamycin (Km), amikacin (Am), capreomycin (CM), viomycin (Vm), streptomycin (S) </li></ul></ul></ul><ul><ul><li>Group 3 </li></ul></ul><ul><ul><ul><li>Fluoroquinolones Moxifloxacin (Mfx), levo (Lfx), Oflox (Ofx) </li></ul></ul></ul><ul><ul><li>Group 4 </li></ul></ul><ul><ul><ul><li>Oral bacteriostatic Ethionamide (Eto), protionamide (Pto), </li></ul></ul></ul><ul><ul><ul><li>second line drugs cycloserine (Cs), terizidone (Trd), PAS </li></ul></ul></ul><ul><ul><li>Group 5 </li></ul></ul><ul><ul><ul><li>Agents with unclear role Clofazimine (Cfz), linezolid (Lzd), Amox Clav, </li></ul></ul></ul>
  41. 54. Policy guidance on DST of second line anti tuberculosis drugs : WHO 2008
  42. 55. Mtb isolated = 393 Total sensitivity performed = 94 MDR TB = 50 isolates XDR TB = 2 isolates Sensitive to all the drugs = 19 isolates
  43. 56. Samples for culture = 1656 Culture positive = 455 MTb = 393 MOTT = 62 DST performed = 94 MDR = 50 XDR TB = 2 R to atleast one drug= 23 S to all drugs = 19 2008: NCR & North India
  44. 58. Reporting TAT for 10 Drugs panel / both 1 st & 2 nd line drugs 10 6 th September 28 th August 9HH0022857 12 19 th August 6 th August 9HH006343 13 9 th August 25 th July 9HG032563 12 17 th July 5 th July 9HG005659 14 15 th July 1 st July 9HG000914 6 26 th August 20 th August 9HH007858 19 24 th June 5 th June 9HF006055 11 2 nd July 20 th June 9HF025442 14 26 th June 11 th June 9HF013364 18 28 th June 9 th June 9HF010873 17 25 th June 7 th June 9HF008253 24 25 27 th May 9HE034724 16 6 th June 22 ND May 9HE028517 TAT in days Reported date Accession date Accession No
  45. 60. Smear examination <ul><li>ZN method </li></ul><ul><li>Kinyoun cold stain </li></ul><ul><li>Auromine O </li></ul><ul><ul><li>Rapid, economical, simple </li></ul></ul><ul><ul><ul><li>Adequate sensitivity in symptomatics </li></ul></ul></ul><ul><ul><ul><li>Overall Low sensitivity (22 – 78% of culture) </li></ul></ul></ul><ul><ul><ul><li>Limit of detection 1 x 10 4 vs 10 2 bacilli/ml </li></ul></ul></ul><ul><ul><ul><li>Species cannot be identified </li></ul></ul></ul>
  46. 61. How to improve microscopy LED Microscopy <ul><li>Fluorescent microscope </li></ul><ul><ul><li>Pro: </li></ul></ul><ul><ul><ul><li>High power rather than oil immersion </li></ul></ul></ul><ul><ul><ul><li>Black background, yellow bacilli </li></ul></ul></ul><ul><ul><ul><li>Increased sensitivity by 10% </li></ul></ul></ul><ul><ul><li>Con: </li></ul></ul><ul><ul><ul><li>High cost, maintenance </li></ul></ul></ul><ul><li>FluoLED Microscopy </li></ul>
  47. 62. Improved diagnostics: Specimen processing <ul><ul><li>Improve yield </li></ul></ul><ul><ul><li>Safer for lab personnel </li></ul></ul><ul><ul><ul><li>Bleach centrifugation </li></ul></ul></ul><ul><ul><ul><li>Flotation method </li></ul></ul></ul><ul><ul><ul><li>Bleach sedimentation </li></ul></ul></ul><ul><ul><ul><ul><li>long contact with bleach will lyse bacilli </li></ul></ul></ul></ul>
  48. 63. Culture based detection Gold standard Slow : weeks Low sensitivity
  49. 64. Improving detection <ul><li>CO 2 production </li></ul><ul><li>O 2 consumption </li></ul><ul><ul><li>Radiometric </li></ul></ul><ul><ul><li>Fluorescent </li></ul></ul><ul><ul><li>Colourimetric </li></ul></ul><ul><ul><li>Pressure sensor </li></ul></ul><ul><ul><li>Decrease TAT by half </li></ul></ul><ul><li>14 C Palmitate  14 CO 2 </li></ul>
  50. 65. Improving detection <ul><li>Alamar Blue </li></ul><ul><li>Microscopic observation of micro colonies (MODSA) </li></ul><ul><li>Bacteriophage </li></ul>
  51. 67. Genotype Mycobacterium test <ul><li>DNA Strip technology: Three steps </li></ul><ul><ul><li>1. DNA isolated from culture </li></ul></ul><ul><ul><li>2. Multiplex amplification with biotinylated primers  230 bp product </li></ul></ul><ul><ul><li>3. Reverse hybridization: </li></ul></ul><ul><ul><ul><li>Chemical denaturation of amplified product </li></ul></ul></ul><ul><ul><ul><li>Hybridization of SS biotin labeled amplicon to membrane bound probes </li></ul></ul></ul><ul><ul><ul><li>Stringent washing </li></ul></ul></ul><ul><ul><ul><li>Addition of Steptavidin/alkaline phosphatase </li></ul></ul></ul><ul><ul><ul><li>AP mediated staining reaction </li></ul></ul></ul>
  52. 69. Genotype Mycobacterium CM/AS
  53. 70. Genotype MTBDR assay
  54. 71. M icroscopically O bserved D rug S usceptibility 24 wells plate (BD), 12 wells used per sample, 4 drug free control, 8 containing drugs Middlebrook 7H9 broth (BD) INH 0.1 & 0.4 ug/ml OACD RMP 1 & 2 PENTA Etham 2.5 & 5 720 ul inoculum used SM 2 & 6 Zip lock, examined from 4 to 15 days daily, A/D 40
  55. 72. MODS 7 days ( 6 to 8) MBBacT 13 (10 to 14) LJ 26 (21 to 33)
  56. 74. Potency of anti-TB drugs against M. tuberculosis Gatifloxacin
  57. 75. What does the clinicians really want ? Is it M. tuberculosis Is it MDR ? What drugs can I use ?
  58. 76. Laboratory Quality standards Turn Around Time (TAT) benchmarks <ul><li>Smear examination must be carried out and reported within 24 hours of receipt of specimen </li></ul><ul><li>Isolation & identification of M. tuberculosis should occur in 14 to 21 days </li></ul><ul><li>The whole process, including susceptibility to first line drugs should be completed in 30 days of specimen receipt. </li></ul>
  59. 77. Immediate goal of any diagnostic mycobacteriology laboratory <ul><li>Identify M. tuberculosis </li></ul><ul><li>Determine Rifampicin resistance </li></ul><ul><li>At least in smear positive cases within 2 weeks. </li></ul><ul><li>Keep a look out for XDR TB </li></ul>
  60. 78. Evidence based Summary <ul><li>Use of rapid identification tests in combination with rapid culture (liquid) has greatly reduced overall turnaround time. </li></ul><ul><li>Use a combination of tests rather than a single test for diagnosis </li></ul><ul><li>It is the system of choice where funding is available </li></ul>
  61. 79. Consequence of this strategy <ul><li>Diagnosis of TB patients : 38% faster </li></ul><ul><li>Initiation of therapy : 4.6 days earlier </li></ul><ul><li>Reduction in mortality : 31% </li></ul><ul><li>Reduction in medical costs: 18% </li></ul>
  62. 80. Take home message <ul><li>Key to reliable results is the </li></ul><ul><li>submission of high quality specimens </li></ul><ul><li>Appropriate infrastructure to handle samples </li></ul><ul><li>Staff training, using SOP & validated tests </li></ul><ul><li>Quality assurance procedures </li></ul><ul><li>Close interaction with clinician and program officers </li></ul>
  63. 81. SRL TB Diagnosis Report card Ideal situation “Aim for the moon” <ul><li>Prompt delivery of specimen to the lab </li></ul><ul><li>Prompt use of rapid, state of the art methods </li></ul><ul><li>Reporting of smear results to specimen submitter within 1 day </li></ul><ul><li>Report of culture identification (Mtb or not) in 21 days </li></ul><ul><li>Report on susceptibility results within 30 days </li></ul>
  64. 82. Diagnosis of TB <ul><li>Current strategy: </li></ul><ul><ul><li>Detection </li></ul></ul><ul><ul><ul><li>Microscopy </li></ul></ul></ul><ul><ul><ul><li>Culture </li></ul></ul></ul><ul><ul><li>Identification </li></ul></ul><ul><ul><ul><li>Biochemicals </li></ul></ul></ul><ul><ul><ul><li>HPLC </li></ul></ul></ul><ul><ul><ul><li>PCR, Probes </li></ul></ul></ul><ul><ul><li>Susceptibility </li></ul></ul><ul><ul><ul><li>First line </li></ul></ul></ul><ul><ul><ul><li>Second line </li></ul></ul></ul><ul><li>Future strategy </li></ul><ul><ul><li>Detection & Identification </li></ul></ul><ul><ul><li>Susceptibility </li></ul></ul><ul><li>Cepheid is NOW </li></ul>24 hrs, 6 wks, 6 months 2 hrs, 24 hrs complete report
  65. 83. If problem is man made, solution too will have to be wo man made Thank you for your attention